J. Goulding1,2, S. J. Mistry3,2, S. J. Briddon1,2, J. Woolard1,2, B. Kellam3,2, S. J. Hill1,2. 1Division of Physiology, Pharmacology and Neuroscience, School of Life Sciences, University of Nottingham, Nottingham, United Kingdom, 2Compare, University of Nottingham, Nottingham, United Kingdom, 3School of Pharmacy, University of Nottingham, Nottingham, United Kingdom.

Introduction: The β2 adrenoceptor (β2AR) is a G protein-coupled receptor implicated in the pathology of asthma and heart disease. Fluorescent antagonists allow both visualisation and study of real-time ligand-receptor interactions in living cells. However few highly selective β2AR fluorescent antagonists are available. Therefore, we have synthesised three new fluorescent analogues of the selective β2AR antagonist ICI-118,551 (1, β2AR pKD 9.3) with varying linkers. We show that these ligands retain β2AR/β1AR selectivity and can be used for single cell ligand binding using Bioluminescence Resonance Energy Transfer (BRET) imaging.

Methods: Three BODIPY630/650-X conjugates of the 3-((3-hydroxy-4-((7-methyl-2,3-dihydro-1H-inden-4-yl)oxy)butan-2-yl)amino core of ICI-118,551 were synthesised with either a 3-(2-(2-(2-aminoethoxy)ethoxy)ethyl- or dipeptidyl (Gly-Ala/β-Ala-β-Ala) linkers. Confocal imaging was performed with a Zeiss LSM880 microscope using HEK293 cells stably expressing an N-terminal SNAP-tagged β2AR (SNAP_β2AR) labelled with 0.5µM SNAPSurface488. Whole cell saturation NanoBRET ligand binding assays were performed on HEK293 cells stably expressing N-terminal nanoluciferase- (Nluc) tagged human β2AR or β1AR as previously reported (2). BRET imaging was performed on Nluc_β2AR cells on an Olympus LV200 bioluminescence microscope using furimazine (4μM) and fluorescent antagonist (100nM). Emission was collected at 438/24nm bandpass filter (20s) and 647nm Long Pass (4min) and image analysis performed using Fiji plugin Time Series Analyzer V3.

Results: Clear saturable specific binding was detected at the β2AR using NanoBRET with all three fluorescent ICI,118551 analogues (Table 1). The polyether linked ICI118,551-BODIPY630/650-X conjugate (PEG3) demonstrated the highest affinity and selectivity for the β2AR (Table 1). Confocal imaging of all three antagonists displayed membrane binding, co-localising with that of the SNAP_ β2AR. Displaceable binding (10μM propranolol) was observed for the Gly-Ala-ICI118,551-BY650/650X conjugate by confocal imaging and by BRET imaging for the β-Ala-β-Ala-ICI118,551-BY650/650X and PEG3-ICI118,551-BY650/650X conjugates.

Linker

β2AR pKD

β1AR pKD

PEG3

7.74 ± 0.05 (6)

<5 (5)

Gly-Ala

7.06 ± 0.10 (5)

<5 (4)

β-Ala-β-Ala

7.36 ± 0.15 (5)

6.32 ± 0.25 (5)

Table 1. Affinity of ICI118,551 derivatives at the human β1 and β2AR in HEK293 cells using NanoBRET saturation binding. Data represent mean ± S.E.M of n repeat experiments performed in duplicate. 10μM propranolol was used to define non-specific binding.

Conclusion: We have developed novel selective β2AR fluorescent antagonists that can be used for confocal and BRET imaging. We thank the MRC for financial support.